Project Summary The ADAMs (a disintegrin and metalloproteases) are transmembrane multi-domain proteins involved in multiple biological events including proteolysis, cell adhesion, fusion, proliferation and migration. Although aberrantly elevated activity of specific ADAMs has been implicated in different diseases, their best-documented role is in tumorigenesis and tumor progression, as a result of their biological functions ? proteolytic ?shedding? of membrane-anchored proteins (e.g., the complete ectodomain of cytokines, growth factors, receptors and adhesion molecules, etc.) and hence the rapid modulation of key cell autocrine and paracrine signaling pathways in the tumor microenvironment. Specific ADAMs that are implicated in oncogenic pathways includes ADAM8/9/10/12/15/17/19/28, among which the strongest evidences for a role in malignance exist for ADAM17 and its closest relative ADAM10. Despite major advances in our understanding of ADAM10/17 during the past decade, numerous questions have emerged regarding their expression in cancer and the mechanisms by which they contribute to tumorigenesis/progression. Substrate identification and discrimination is critical to shed light on the potential mechanisms, but little effort has focused on the identification of substrates of ADAMs, in part because of where they localize?the cell membrane. Here, we propose to develop a high-throughput method to comprehensively profile protease substrates of ADAM10/17 using phage display and massively parallel DNA next generation sequencing (NGS). By constructing two phage libraries displaying either randomized 10-mer peptides or 49-mer human protein segments covering the entire human proteome, we will be able to compare proteolysis specificities for peptides versus native human proteins, getting insights in how folding and domain structures dictate the accessibility of the cleavage site. Using this approach, we intend to define the cleavage site selectivity of ADAM10/17 proteases. In addition to conducting in vitro screening with soluble forms of ADAM10/17, which might be biased due to the location, orientation and conformation constraints imposed by their role as ecto-proteases, we will perform on-cell selection by engineering the displayed peptides on phages to have it insert into cell membranes, mimicking landscapes of membrane substrates. By identifying and characterizing the functional importance of the substrates of ADAM10/17, we hope to unveil more information regarding the roles of ADAM10/17 in cancer development and validate the hypothesis that pharmacologically targeting ADAMs would be useful. Finally, we will develop recombinant antibodies selectively targeting active forms of ADAM10/17 in hope to inhibit ADAMs involved oncogenic signaling. In the long term, this technique should be useful for studying the sequence specificity of a variety of other posttranslational modifications (PTM), including phosphorylation, citrullination, and sulfonation.